KR101649375B1

KR101649375B1 - The method of manufacturing the transplantable spheroids of mixed cellular complexes for cell transplantation and the usage of the same

Info

Publication number: KR101649375B1
Application number: KR1020090089912A
Authority: KR
Inventors: 이정익
Original assignee: 건국대학교 산학협력단
Priority date: 2009-09-23
Filing date: 2009-09-23
Publication date: 2016-08-30
Also published as: KR20110032433A; WO2011037416A2; WO2011037416A9; WO2011037416A3

Abstract

[assignment]

The present invention provides a clinically effective and practically usable cell spleen for transplantation therapy to treat transplantation of a part or all of the damaged or defective lesion in a disease state out of the normal state, The present inventors have confirmed that the present invention can be used as a system for evaluating the drug efficacy and toxicity of the test substance and a method for treating each disease using the present invention which can substitute various tissues constituting the body expressing the transcript as a tissue similar to the condition And has reached the completion of the invention.

[Solution]

According to one aspect of the present invention, there is provided a method for producing a mixed cell complex, comprising the steps of: (1) preparing each cell material constituting a mixed cell complex; (2) subculturing the isolated cells to amplify them; (3) preparing a cell material suspending liquid in which one or more kinds of cells are singly or mixed in a culture medium, and culturing the cells in a high density suspended state to prepare a mixed cell complex by adhering the cell materials to each other ; And (4) separating the cell spleoid and transferring the cell spleoid to a predetermined place according to the purpose of use for each purpose, and a method of using the same.

Pancreatic endocrine cells (PECs), which are isolated from cells, are cultured in the presence or absence of cells, such as cell spleens, chondrocytes, synovial cells, mesenchymal stem cells, autologous chondrocyte implantation, adult stem cells, , Endothelial progenitor cells (EPC)

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing a cell-spleen, which is a mixed cell complex for cell transplantation, and a method of using the same,

The present invention is based on the finding that the mixed cell complex has a problem in balance of homeostasis of magnetic tissue regenerative therapy ability due to hereditary diseases, infectious diseases and degenerative diseases, A group of cells capable of replacing various tissues constituting a body expressing a transgene as a tissue similar to a normal or healthy state in the body, The present invention relates to a method for producing a cell spoloid, and relates to a field of biology, medicine, etc. of each tissue in each animal body.

The mixed cell complex is a tissue similar to a normal or healthy state in the body, and can be used in a large number of cells that can replace various tissues constituting the transgene expressed in the body (for example, in cartilage tissue, Each cell material composed of cell spleoid and cell spleoid expressing as islet endocrine cells that secrete insulin and the like in pancreatic tissue was prepared, each cell prepared for separation was subcultured and amplified, and then one kind Alternatively, two or more kinds of cells may be singly or mixed in a culture medium, and the cell material suspension may be prepared and shaking culture may be carried out to prepare a mixed cell complex by adhering the cell materials to each other. Method for the preparation of cell spleens for graft treatment, comprising the step of transferring the cells to a predetermined site according to the method It relates to a method.

Cell therapy transplantation is increasingly being used as a means of research in the academic field and as a tool for technology development in the industrial field. These cell therapy transplantations are becoming more and more important in the national industry as they have a large ripple effect on various industries.

Cell therapy for use in cell therapy transplantation (cell therapy product) is incubated in a living self (autologous), allogeneic (allogenic), or two kinds (xenegenic) in vitro environment (in vitro) the cell in order to restore the function of cells and tissues Refers to a drug used for therapeutic, diagnostic, and prophylactic purposes through a series of actions that separate, proliferate, screen, or otherwise alter the biological characteristics of a cell through a process.

Cellular therapy using a cell therapy agent Each cell material, which is similar to a normal or healthy state in the body used in transplantation, is composed of a plurality of cells capable of replacing various tissues constituting the expressed body, , A series of actions that change the biological characteristics of a cell through a culturing process in the in vitro environment is applied to the recipient to be implanted, or the tissue is used to treat human tissue.

Since cell therapy agents used in cell therapy transplantation are cell units, they can be used to newly retain specific functions (eg, secretion of hormones, recognition of specific antigens, etc.), genetically modified cells And these cell therapeutic agents can be classified into an adult cell therapeutic agent and a stem cell therapeutic agent depending on the degree of differentiation of the cell used.

Examples of the adult cell therapeutic agent include autologous chondrocytes or bone cells used for autologous chondrocyte implantation for treating cartilage tissue and bone tissue damage, islet cells for treating diabetes, various cardiovascular diseases, and diseases caused by impaired peripheral vascular system Endothelial progenitor cells (EPC), endothelial stem cells and cardiac muscle cells and muscle cells, epidermal cells for skin regeneration, fetal nerve cells for the treatment of Parkinson's disease natural killer cells, dendritic cells and cytotoxic T cells for the treatment of various dopaminergic, cancer and immune diseases, including corneal epithelial cells, fetal neural cells, And corneal cells. Cells derived from various organs or tissues constituting the body can also be used as agents for treating adult cells. These cells may be aggregates or sections of a plurality of micro cells obtained by chopping organs or tissues to be transplanted have.

On the other hand, the stem cell treatment agent is similar to embryonic stem cells (ES cells) in terms of using stem cells derived from various tissues showing a high degree of undifferentiation and having a multi-potency and high proliferation capacity Functional and proliferative ability and has a similar ability to a normal or healthy state in the body and has an easily differentiating ability and is transplanted into a damaged area to show a therapeutic effect, thereby being able to replace one or more kinds of tissues constituting the inside of the body Two or more kinds of cells are separated and differentiated into desired cells, and the resultant cells are used.

Cell therapy used in cell therapy transplantation can be used to directly regenerate the function or tissue of a damaged cell by injecting cells directly into the patient, so that it is not toxic in the body and regenerates and maintains the original function of the body tissue, Or radical therapy that can not be done with pharmacotherapy.

Typical diseases that can be treated with such a cell therapy agent and cells used therein include autologous chondrocytes or bone cells used for autologous chondrocyte cell transplantation for treating damage to cartilage tissue and bone tissue, Endothelial progenitor cells (EPC), endothelial stem cells and cardiac myocytes and muscle cells for treating diseases caused by diseases such as cancer, cell, various cardiovascular diseases and peripheral vasculature. In addition, it is as follows.

Countries such as Korea, Japan, and Europe are already facing an aging society, and their average life expectancy is becoming the highest in the world. People's hopes began to focus on quality of life (QOL) so that they could live more satisfactorily than simply life extension. One of the noteworthy of these is the impairment of motor function.

In 2002, more than 70 million patients in the United States visited the medical facility for arthritis-associated diseases or chronic joint symptoms, although arthritis, which is a cause of motor function disorder, includes various diseases. This is one in three adults, and by 2020, it is expected to double the number of patients.

The proportion of patients who experience discomfort in their daily lives is second only to cardiovascular disease, followed by medical expenses of $ 86.3 billion a year. Osteoarthritis (Osteoarthritis) is one of the leading causes of aging society, and it is estimated that 24 million people will be bypassed in a survey of 22 Century Medical Center of Tokyo University of Japan. In Japan, the prevalence of patients is high, ranging from 45 to 65 to 30 percent, and from 65 to 65 percent, with a prevalence of 63 to 85 percent, with an estimated 900,000 new cases occurring annually. Degenerative arthritis is a typical athlete's disease, unlike a real organs disease, but directly threats the life, but the human hand and feet to take away the freedom to significantly lower the QOL. These diseases are expected to increase more and more by the aging society in the future, and human and social losses caused by these disorders are considerable.

Most of these exercise disorders are caused by inflammation or damage of cartilage tissue and bone tissue. In the present case, severe diseases are treated with artificial joints made of metal and polyethylenes of high molecular weight. However, when it is over 10 years after replacement with artificial joints, And to cause a reaction. In order to solve these problems, researches have been carried out to make the wear resistance constant, but there is a limit in wear resistance.

In 1994, Brittberg et al. Reported the use of autologous chondrocyte implantation (Brittiberg et al., 2002) for the transplantation of cartilage tissue cells isolated from the mid- New England Journal of Medicine, 331 (14), 889 (1994)), and has been approved by the FDA in 1997 and has already been commercialized and performed for more than 20,000 cases worldwide. The median and long-term results of 219 patients were good and 89% were functional improvement (2, 10 years) (PETerson L, 6 ^th Annu., American Academic Orthopedic Surgery (1998)). In 2002, there were reports of deaths due to bacterial infection after transplantation. According to the CDC survey, 41 cases of infection were confirmed. In Japan, the Japanese Ministry of Health, The fact that the information was provided reaffirms that there are issues that need to be rethinked carefully. In addition, this method can not be used for degenerative arthritis accompanied by extensive cartilage tissue and degeneration or partial defects of the bone tissue, which are frequent in the elderly, and still needs to be improved.

In Japan, a cartilage tissue isolated from non-midcraft articular cartilage or mesenchymal stem cells derived from bone marrow has been used to produce cartilage tissue and clinical applications have been started in the osteochondral defect. However, A case of traumatic osteochondral injury or herniated osteochondritis has been limited to the application of a limited range of cartilage defects from the beginning (Japanese Patent Laid-Open Publication No. 2001-384446 (JP-A-2003-180819), JP-A 2002-216561 (Japanese Patent Application Laid-Open No. 2003-111831), Japanese Patent Application Laid-Open No. 2003-58118 (Japanese Patent Application Laid-Open No. 2004-136096)). At present, it has not been used for treatment of degenerative arthritis with degenerative or partial defect of cartilage tissue and bone tissue because it has obtained stable therapeutic result with artificial joint replacement. Most of these techniques are cartilage tissues and bone tissues in which most of them are made of a single kind of cells (i.e., cartilage cells alone, osteocytes alone or stem cells alone), as well as proteins produced from cultured cells, Since a substrate for tissue regeneration, which is a support made of a protein, a saccharide or an artificial biomaterial, which is a substance for preventing the single cell suspension to be grafted from being detached from the transplantable part, is supplementarily or supplementally used, A direct effect of a living body due to defects such as foreign body reaction and inferior fitness in vivo has to be solved. Therefore, in reality, it is considered that the development of a technique for minimizing the use of such a support is desperately needed in view of such problems that clinical application is not practical.

On the other hand, Hunziker et al., Which is actively performing the treatment of degenerative arthritis from the basic research point, is concerned that the condition of degenerative arthritis is degeneration of the cartilage and damage of the cartilage tissue, Based on the partial defect model. In this study, we have shown that the central role of cartilage repair and regeneration is not synovial cells but synovial cells (Hunziker et al., The Journal of Bone and Joint Surgery, 78-A, 721 (1996)). However, the limited therapeutic range of this technique is also considered to be a practical treatment for widespread degeneration, which is unrealizable and is at an early stage in establishing early therapeutic techniques for a wide range of osteochondral defects.

The presence of adult stem cells was confirmed in each tissue in the body, and the mesenchymal stem cells derived from various tissues were also found localized. In particular, synovial cells derived from synovial cells and bone marrow derived cells are differentiated into tissues expressed as cartilage sheath tissues by using mesenchymal stem cells having differentiation ability into chondrocytes, and they are reconstituted in an in vitro and in vivo environment A number of studies have been reported that have succeeded. Adult stem cells are able to differentiate and regenerate into various tissues constituting the living body. Especially, the cells are highly proliferative compared to somatic cells derived from general tissues by culturing in vitro. It is considered that mesenchymal stem cells having such a high proliferation rate are advantageous from the viewpoint of embryogenesis or differentiation. On the other hand, Sekiya et al. Have confirmed the excellent regeneration and restoration of cartilage defect sites in vitro and in vivo using cells derived from synovium (Sekiya et al., Stem cell, 25, 689 (2007)). Chen et al. Have found that synovial-derived cells co-cultured with bone marrow cells resulted in differentiation into cartilage tissue (Chen et al., Spine, 34-12, 1272 (2009)). Anderer et al. Have reported that, in order to differentiate into cartilage tissue, only the culture medium used for ordinary cell culture, without using the culture medium for inducing cartilage differentiation, which is formed by addition of proteins such as growth factors as well as proteins produced from cultured cells, Cartilage regeneration has been successful (Anderer et al., Journal of Bone and Mineral Research, 17-8, 1420 (2002)).

On the other hand, diabetes is largely divided into 'insulin dependent diabetes' and 'insulin independent diabetes', but the precise mechanism of diabetes that differentiates the two types of diabetes is not yet known It is not revealed. Of the two types of diabetes, insulin-dependent diabetes mellitus requires insulin to be continuously supplied from the outside because the ability to produce insulin is greatly disturbed. However, it is almost impossible to supply insulin continuously in accordance with the physiological demands. Further, insulin concentration gradient exists in the body, and the concentration of insulin decreases in order of 'hepatic vein → liver → hepatic vein → aorta → muscle' When insulin is externally injected into the body, such concentration gradients are not formed and side effects occur.

In the pancreatic beta-cells, besides insulin, eleven kinds of other substances are secreted together to maintain metabolism smoothly. Therefore, insulin alone can lower blood glucose, but it is impossible to prevent hypoglycemia and other complications. In addition, the hypoglycemic agent used for the treatment of diabetes has a hypoglycemic effect, but it is not used because it is difficult to use for a long period of time due to a hypotensive agent resistance and a serious side effect. As an alternative to this, islet transplantation is a new treatment for severe insulin-dependent type 1 diabetes. In recent years, clinical treatment reports have been increasing every year in the world because of the fact that they can be treated without restenosis, such as renal insufficiency, retinopathy, and neurotoxicity, which can not be solved only by administration of insulin.

(2001), Lee et al., Cell Transplantation 17, 51, (2008)), for the purpose of eliminating the immune rejection and transplanting the islets in different ways.

On the other hand, the inventors of the present invention have developed microcapsules for encapsulating the islets to be transplanted using elastic cartilage derived from an individual to receive the transplants for immuno-sequestration (Korean Patent Registration Document 10-0788800 (WO / 2008/002059)).

However, at present, the supply of islets to be transplanted is absolutely insufficient. Thus, in a normal state, endocrine cells isolated from a single cell by a predetermined treatment in an islet, which is a mixed cell complex containing various kinds of endocrine cells, endocrine cell (PEC)), and methods that can be used as a treatment for cell transplantation through the production of transplantation cell spoloids comprising the above-described mass proliferated cells Can be considered.

In addition, the formation of cardiovascular system is one of the phenomena occurring at an early stage in the developmental stage, and even after becoming an adult, it has the same dynamic structure repeatedly emerging, restoring and dying throughout life. Recently, there has been a remarkable development of stem cells or regenerative medicine, as well as cells that play an important role in angiogenesis while performing active treatments for various tissue damage in vascular restoration after adulthood as well as in the early development stage .

Endothelial progenitor cells (EPCs), one of the monocyte components in peripheral blood, proliferate and differentiate in the bone marrow tissues responsible for angiogenic angiogenesis, migrate to the site where angiogenesis is in progress, (Asahara et al., Science 275, 964, (1997), Asahara et al., EMBO J, 18, 3964 (1999), Takahashi et al., Nature Medicine, 4,434 (1999)). Currently, in clinical trials, vascular regenerative therapy by vascular endothelial progenitor cell transplantation therapy has been attempted for coronary artery disease, ischemic disease, and severe ischemic nitrification (JP Patent Publication No. 2005-286607 (Tokukai 2007-89537 )). Considering these factors, blood and endothelial progenitor cells are expected to play a role in regeneration of cardiovascular and various tissues and organs. In order to more aggressively apply vascular endothelial cells to the clinic, it is necessary to quantitatively and functionally improve blood and endothelial progenitor cells present in an extremely small amount.

The movement to apply vascular endothelial progenitor cells to vascular regenerative therapy can be explained by several excellent points that these vascular endothelial progenitor cells possess than embryonic stem cells or other tissue-specific adult stem cells. In addition to being able to be used for the tissue regeneration of the patient using his or her blood or bone marrow tissue, it is due to the unique therapeutic intrinsic properties of EPC. Vascular endothelial progenitor cells have been shown to secrete large amounts of vascular regeneration factors, such as 1) VEGF (Vascular Endthelial Growth Factor), Angiopoietin-1, and HGF, which do not frequently develop into other cell types 3) it actively acts on angiogenesis in tissue, and 4) the relatively short half-life of transplanted cells is the best cell for clinical treatment of cardiovascular diseases through blood vessel regeneration.

The disease state deviating from the normal state is an incomplete disease state of the parenchyma due to various cardiovascular diseases and peripheral vascular system failure. Especially in the body, since the vascular system is distributed in almost all tissues, One of the diseases is the regeneration of new blood vessels, which requires the reconstruction of a healthy cardiovascular system. In this case, the vascular endothelial cells, endothelial progenitor cells (EPC), endothelial stem cells and cardiac muscle cells Or endothelial cells in various tissues in the body which require the induction of neoangiogenesis, such as the recruitment of endothelial cells (e. G., Muscle cells) progenitor cells (EPCs), endothelial stem cells and cardiac muscle cells and muscle cells, and the like. Through the preparation of a therapeutic cell spheroid may want to consider the methods that can be used in the treatment of cell transplantation.

Taking into consideration the above results, the present inventors have found that the production of cell spleens for transplantation therapy, which is a mixed cell complex, with mesenchymal stem cells, pancreatic endocrine cells (PEC) isolated from a pancreatic islets, and endothelial progenitor cells And the present invention has been accomplished.

Particularly, under normal cell culture conditions, mesenchymal stem cells, pancreatic endocrine cells (PEC) and endothelial progenitor cells isolated from a pancreatic islets are highly proliferative, and cartilage tissue, pancreatic tissue and blood vessels Having an ability to easily differentiate into tissues makes it possible to produce a desired amount of cell spleen for transplant therapy within a short period of time.

Recently, a number of techniques have been reported to apply cells or tissues cultured and cultured in vitro in patients. In this case, a cell or tissue of one kind may be used as a structure for regenerating a tissue or a culture solution or a solution for transportation, which is a scaffold formed of a biocompatible material or a bioabsorbable material such as an artificial biomaterial, (Langer et al., Science 260, 920, (1993)) and regenerative medicine (Petit-Zeman, Nature Biotechnology 19, 201, (2001)) that regenerate tissues in vivo A tissue similar to a normal or healthy state in the body including the treatment of bone tissue, the treatment of diabetes, and the treatment for diseases caused by various cardiovascular diseases and peripheral vascular diseases, Therapeutic treatments have been attracting attention by making human tissues.

In the meantime, autologous chondrocytes or bone cells used for autologous chondrocyte transplantation for treating the damage of cartilage tissue and bone tissue developed by tissue engineering and regenerative medicine, islet cells for diabetes treatment, various cardiovascular diseases, A transplantable body prepared in an in vitro environment such as vascular endothelial cells, vascular endothelial progenitor cells, endothelial stem cells and cardiac muscle cells and muscle cells for treating a disease state due to peripheral vascular system failure, a tissue similar to a normal or healthy state , The regenerated tissues expressing the transgene and the related graft cells constituting the structure were mostly made of a single kind of cells.

The treatment for cartilage tissue and bone tissue during treatment, treatment for diabetes, and treatment for diseases caused by various cardiovascular diseases and peripheral vascular system disorders include autologous chondrocytes or bone cells used for autologous chondrocyte transplantation cultured in the affected area, (PEC), endothelial cells, endothelial progenitor cells (EPC), endothelial stem cells and cardiac muscle cells and muscle cells, or individual tissues made of these cells, The method of implantation can be considered.

[Prior Art Literature]

[Non-Patent Document]

[Non-Patent Document 1] Langer et al., Science 260, 920, (1993)

[Non-Patent Document 2] Petit-Zemane, Nature Biotechnology 19, 201, (2001)

[Non-Patent Document 3] Brittiberg et al., New England Journal of Medicine, 331 (14), 889 (1994)

[Non-Patent Document 4] Peterson L, 6 ^th Annu. Meet., American Academic Orthopedic Surgery (1998)

[Non-Patent Document 5] Hunziker et al., The Journal of Bone and Joint Surgery, 78-A, 721 (1996)

[Non-Patent Document 6] Sekiya et al., Stem cell, 25, 689 (2007)

[Non-Patent Document 7] Chen et al., Spine, 34-12, 1272 (2009)

[Non-Patent Document 8] Anderer et al., Journal of Bone and Mineral Research, 17-8, 1420 (2002)

[Non-Patent Document 9] Shapiro et al., Lancet 358 Suppl, S21, (2001),

[Non-Patent Document 10] Lee et al., Cell Transplantation 17, 51, (2008)

[Non-Patent Document 11] Asahara et al., Science 275, 964, (1997),

[Non-Patent Document 12] Asahara et al., EMBO J, 18, 3964 (1999)

[Non-Patent Document 13] Takahashi et al., Nature Medicine, 4,434 (1999)

[Patent Literature]

[Patent Document 1] Japanese Patent Registration Document: JP-A-2003-180819

Report

[Patent Document 2] Japanese Patent Registration Document: JP-A-2003-111831

Report

[Patent Document 3] Japanese Patent Registration Document: Japanese Patent Application Laid-Open No. 2004-136096

Report

[Patent Document 4] Korean Patent Registration Document: Patent No. 10-0788800

[Patent Document 5] Japanese Patent Registration Document: JP-A-2007-89537

Report

The present inventors have found that, in order to provide clinically effective and practically usable cell spleens for transplantation treatment of mixed cell complexes, the mixed cell complexes can be classified into various types of tissues constituting the body expressing the transgene as a tissue similar to a normal or healthy state in the body The cell material constituting the cell spoiloid and the mixed cell complex, which is a collection of a plurality of cells capable of replacing the tissue, is prepared, the separated cells are subcultured to be amplified, and then one or two or more kinds of cells Cellular material complexes are prepared by adhering cell materials to each other by preparing shake culture of single or mixed cell material in the culture medium, separating the prepared cell spoloids, And a method for preparing a cell spleroid for transplantation therapy comprising the steps of: And a system for evaluating the physiological action or toxicity of a drug substance and toxicity of a test substance such as a compound, a drug, and a toxic substance separately from the purpose of treatment.

When applied to autologous chondrocyte transplantation to treat damage to cartilage tissue and bone tissue, each cell material constituting the cell spoil for transplantation treatment is used not only for the sole use of autologous chondrocytes or bone cell chondrocytes, Or a variety of stem cells or cartilage precursor cells having two or more kinds of highly proliferative ability and having easy differentiation ability into cartilage tissue can be used to prepare a large amount of cell spoiloids for transplantation therapy And thus it is very useful in terms of the establishment of a treatment technique for a wide range of cartilage tissues and bone degeneration or degeneration of cartilage and damage of cartilage tissue to the elderly.

In addition, each cell material constituting the cell-spleen for transplantation treatment may be a pancreatic endocrine cell (PEC) isolated from a pancreatic islet cell or an islet cell for the treatment of diabetes, or a variety of cardiovascular diseases and peripheral vascular diseases Endothelial progenitor cells (EPC), endothelial stem cells, and cardiac myocytes and muscle cells for the treatment of disease states caused by islets and vascular tissues , It is possible to prepare a large amount of cell spoiloids for transplantation therapy expressing the transgene as an alternative to the situation that the supply of the transplanted islets is insufficient due to the chronic shortage of the organ in the clinical pancreas transplantation, It is not a cell suspension consisting of a single cell for transplantation therapy but one or more three-dimensional structure By using the cell spoiloid, which is a collection of multiple cells, it is possible to further promote the specific function originally possessed and to communicate closely with each constituent cell in the structure of cell spoiloid without distributing the cells after transplantation It is very useful in terms of In addition, it may be useful to have new functions such as immunosuppression depending on the characteristics of the cells used.

The present invention aims to provide a method for producing the cell spleen for transplantation and a method for using the same.

In one aspect, the present invention provides a mixed cell complex comprising a cell spleen for graft treatment, which is a collection of a plurality of cells capable of replacing various tissues constituting the body expressing the trait as a tissue similar to a normal or healthy state in the body Lt; / RTI >

In another aspect, there is provided a method for preparing a mixed cell, comprising: (1) preparing each cell material constituting a mixed cell complex; (2) subculturing the isolated cells to amplify them; (3) A cell material suspension prepared by mixing one or more kinds of cells alone or in combination in a culture medium is prepared, and the cells are maintained in a high-density floating state to produce a mixed cell complex step ; And (4) separating the cell spleen and moving the cell spleoid to a predetermined place according to each use method for each purpose.

In the present invention, the cell spoloid for grafting therapy may be used in the autologous chondrocyte transplantation to treat damage to the cartilage tissue and bone tissue of each cell material constituting the mixed cell complex, In addition to being used alone, it has various types of stem cells or cartilage precursor cells which have high proliferative ability of one kind or two or more kinds and have easy differentiation ability into cartilage tissue. Thus, It is possible to prepare a large amount of lloids and it is also very useful in terms of establishing a therapeutic technique for a wide range of cartilage tissues and bone tissue degeneration or degeneration of cartilage and loss of cartilage tissue which do not reach the cartilage of the elderly It is means. In addition, since the present invention does not use a periosteal patch, it is possible to reduce the burden on the patient side compared to conventional autologous chondrocyte grafting, minimize the incision site, and reduce invasion by surgery.

On the other hand, the inventors of the present invention have conducted studies on the preparation of a carrier suitable for cartilage regeneration by tissue engineering techniques, studies on the construction of an intelligent extracellular environment, studies on cartilage regeneration by allograft of cartilage manufactured by tissue engineering, And research on cartilage repair or regeneration by cartilage cell sheet or regenerated cartilage plate without using a support as artificial material. These results suggest that the presence of mesenchymal stem cells mobilized to the injured area is a prerequisite for the treatment of damage and that the presence of tissue-engineered cartilage components as an initiator of minimal cartilage induction in tissue repair and regeneration is important. I am suggesting.

Considering the opinion of the present inventors, the partial defect that the cartilage tissue layer does not reach from the cartilaginous bone has a pluripotent and proliferative capacity, and the mobilization of the mesenchymal stem cells having the ability to differentiate into cartilage tissue is small, There are many cases where only incomplete treatment with insufficient restoration and regeneration is not expected. The cell spleen for transplantation prepared by using the mesenchymal stem cell and the chondrocyte as the mixed cell body of the present invention, such as synovial-derived or bone marrow-derived, satisfies the above-mentioned condition, and therefore, the result of good tissue restoration and regeneration can be expected .

In addition, each cell material constituting the cell-spleen for transplantation treatment may be a pancreatic endocrine cell (PEC) isolated from a pancreatic islet cell or an islet cell for the treatment of diabetes, or a variety of cardiovascular diseases and peripheral vascular diseases Endothelial progenitor cells (EPC), endothelial stem cells and cardiac myocytes and muscle cells for massive proliferation to treat disease states caused by islets and vascular tissues The present invention provides a large amount of preparation of cell spleen for transplantation in which the transplantation of the transplantation is performed, and it is an alternative to the fact that the supply of the transplanted islet is insufficient due to the chronic shortage of the organ in clinical islet transplantation, It is not a cell suspension consisting of a single cell for therapy, but one or more three-dimensional structures By using the cell spoiloid, which is a collection of multiple cells, it is possible to further promote the specific function originally possessed and to communicate closely with each constituent cell in the structure of cell spoiloid without distributing the cells after transplantation It is very useful in terms of In addition, it may be useful to have new functions such as immunosuppression depending on the characteristics of the cells used.

The present invention relates to a mixed cell complex in which a mixed cell complex is a cell for transplantation therapy, which is a collection of a plurality of cells capable of replacing various tissues constituting the body expressed as a tissue similar to a normal or healthy state in the body As a steroid, it is possible to use as a prophylactic or therapeutic agent for the treatment or prevention of arthritis, arthrosis, cartilage damage, cartilage bone defect, meniscus injury, disc degeneration or degenerative arthritis (Osteoarthritis) And the like. The present invention also relates to a cell spleen for transplantation therapy for the treatment of an incomplete disease of the parenchyma due to various cardiovascular diseases and peripheral vascular system failure.

In the present invention, the term "mixed cell complex" means a cell spoil which is a group of cells capable of replacing various tissues constituting the body expressing the trait as a tissue similar to a normal or healthy state in the body, A progenitor cell and an oocyte of a final differentiation stage exhibiting a unique function derived from the donor tissue, a progenitor cell and an oocyte of the adult cell, and an adult stem derived from various tissues showing a high degree of undifferentiation, a multi-potency, It has pluripotent and proliferative capacity similar to ES cells, such as stem cells of mesenchymal stem cells, embryonic stem cells (ES cells), and inducible pluripotent stem cells (iPS cells) And has an easy differentiation ability, and is implanted into injured lesion of cartilage tissue, islet tissue, or vascular tissue And may be a cell spoloid characterized by being a collection of a plurality of cells composed of one kind or two or more types of cells capable of replacing various tissues constituting the inside of the body by showing a therapeutic effect. This is because the pluripotent cells are undifferentiated cells and are expected to differentiate into cells suitable for the biotissue of the transplantation site after transplantation.

It may also be a cell spleod consisting of cells derived from a collection or section of a plurality of micro-units of cells obtained by mincing organs or tissues to be transplanted from a normal or healthy state in the body.

Mixed cell complexes are preferably cell material derived from female tissue for ideal cell transplantation without immunological rejection or ethical problems, but cartilage tissue does not have blood vessels, nerves, and lymphatic vessels under normal conditions, Since it is known that it is an immune-privilege having low immunogenicity that is difficult to induce an immunological rejection reaction at the time of transplantation, it is known that a cell derived from a homogeneous or heterogeneous tissue or two or more of these cells Cell.

In the present invention, each cell material derived from each of the corresponding donor tissues that can be selected in accordance with the purpose of use is not particularly limited. As an embodiment, in addition to cartilage cells as adult cells in a final differentiation stage exhibiting an inherent function, in the case of a cell which is responsible for an endocrine function, an excellent effect for treating a specific disease can be expected. For example, if the graft is derived from the pancreas, it can be used to treat diabetes, and if the graft is derived from the thyroid gland, it can be used to treat hypothyroidism, respectively. In another example, the transplant may be used to treat anemia, dwarfism, and hemophilia when the cells secrete erythropoietin, cells that secrete growth hormone, or cells that secrete blood clotting factors, respectively. In another embodiment, the cell line can be used as a system for assessing the therapeutic efficacy or toxicity of the test substance, even if the cell line is a commercially available established cell line in which the information is publicly available and can be purchased or transferred through the related organization.

In the present invention, it is preferable that the cell spoil of a mixed cell body for transplant treatment has a size in the range of 10 mu m to 1,500 mu m. It is preferable that the cell-spleroid for transplantation treatment is about 600 탆 in size. The spleroid produced from somatic cells derived from normal tissues can be cultured under the usual culturing conditions until the distance from the center of the spleen to the surface layer of spoil is about 300 탆 Gas and nutrient confinement becomes more smooth, which makes it possible to prolong the life span of the cell spheroroid in vitro and to promote the proliferation, which is much more advantageous in exerting the original function of the implant. The relatively thin-walled mixed-cell complex is able to spread the gas and nutrients more smoothly, thereby extending the lifespan of the graft and enabling the expression of the trait as cartilage tissue to be maintained, thereby exerting the original function of the cell spheroid Because it works favorably.

On the other hand, the cartilage tissue is abundant in the extracellular matrix secreted from the cartilage cells in the normal state, and the cartilage cell itself is only a few percent. In addition, when chondrocytes with very low metabolism are in the form of spheroids of the mixed cell complex, the distance from the center of the spheroid to the surface layer of the spheroid, which is capable of diffusion of good gas and nutrients as compared with other cells, is more than 300 μm But is preferably shorter than 1,500 [mu] m.

The present invention provides a method for preparing a mixed cell complex, comprising the steps of: (1) preparing each cell material constituting a mixed cell complex; (2) subculturing the isolated cells to amplify them; (3) preparing a cell material suspending liquid in which one or more kinds of cells are singly or mixed in a culture medium, and culturing the cells in a high density suspended state to prepare a mixed cell complex by adhering the cell materials to each other ; And (4) separating the cell spleens and transferring the cells to a predetermined place according to each purpose of use.

The following manufacturing method according to the present invention will be described specifically by taking as an example a case where each cell material constituting the cell spoil is applied to autologous chondrocyte implantation for treating damage to cartilage tissue and bone tissue.

In the manufacturing method step (1) of the present invention, each cell material constituting the mixed cell complex is prepared separately. First, the corresponding donor tissue, which is the origin of each cell material constituting the mixed cell complex, is sampled to remove skin tissue, subcutaneous tissue, muscle tissue, subchondral bone, ligament, meniscus and other connective tissues Chopped and chopped finely by physical means such as syringe, syringe, forceps, ultrasound device. At this time, a substrate such as a base material made of a resin material including a culture dish, a centrifuge vessel, etc., a watch glass and the like is used as an aseptic base material for accommodating the provided tissue , And if the provided tissue is a flexible tissue such as cartilage tissue, the chipped tissue may be scattered from the accommodating substrate in which it is chipped. This phenomenon occurs when the corresponding donor organization reduces churn of the tissue after it is chopped, and if chopping is carried out by returning the dislodged tissue scattered from the accepted placement, The possibility of contamination due to fungi and the like is increased, and it becomes difficult to smoothly carry out the intended chopping. As described above, in the case of the primary culture including the step of finely carving and preparing each cell material from the provided tissue, there is a fear of contamination by bacteria or fungi. Therefore, in order to prevent contamination by these bacteria, This is because work is required.

The target tissue is placed on a floor in a centrifuge vessel having a concave shaped bottom and a side length sufficient to prevent scattering from the receiving substrate on which the target tissue is located during chopping, curved scissors). The commercially available centrifuge container having a capacity of 50 ml has such a side length as to be able to prevent scattering of the larval tissue from the centrifugal container during chopping so that even when it is a flexible tissue such as a cartilage tissue, It does not escape from the space inside the container.

In addition, when this method is used, the surgical scissors are curved and it is easy to make close contact with the concave bottom of a commercially available 50 ml centrifuge container used for cell culture. Since the target tissue is usually affected by gravity, there is always a tendency to be positioned toward the concave bottom. In this situation and the concave bottom of the centrifuge container, the cutting motion by the surgical scissors causes the tissue to be gathered on the floor, and the cutting motion of the surgical scissors is strongly acted on the elastic cartilage, Even a resilient tissue such as a tissue can be finely chopped to a smaller size in a short period of time. In order to easily accomplish such chopping, it is preferable to perform chopping using the curved surgical scissors longer than the length of the major axis of the side surface portion of the centrifuge container.

A thermostatic water tank capable of providing hot water set at a temperature equal to the body temperature of the individual after being chopped and finely pulverized and then treated with at least one protease selected from neutral protease, trypsin, serine protease, elastase and collagenase In a cell suspension composed of the provided tissue and a protease solution under the environment of air such as a cell culture incubator capable of providing air set in the water or the same temperature as the body temperature of the individual It is digested while causing gait.

Temperature and time to process the protease in the present invention is the electronic scan attack under the conditions of, but can vary depending on the species of the type and the objects of the protease, 37 ℃ in the incubator for a conventional cell culture, 5% CO ₂ ( Magnetic Stirrer).

The reason can be explained as follows. In the process of digesting with proteolytic enzymes, this method uses a conventional incubator for cell culture, an aseptic glass container, and a sterilizing magnet bar for an electronic stirrer. Such a cell culture container material is kept clean, chopped into an aseptic glass container, It is easy to induce gait by flow of liquid motion in cell suspension composed of the provided donor tissue and proteolytic enzyme solution, and the lid of the glass bottle is opened a little so that the gas such as the temperature and humidity of the space inside the incubator The gas composition inside the incubator, which is directly connected to the composition and ideally adjusted for cell culture, is stable and advantageous for each cell in the cell suspension composed of the provided tissue and proteolytic enzyme solution.

In the production method step (2) of the present invention, the separated chondrocyte and synovial membrane-derived cells are subcultured and amplified. Here, as the medium, any cell growth medium or cartilage differentiation induction medium known in the art can be used, and the components (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins) (D-MEM), Minimum Essential Medium (MEM), RPMI-1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 -MEM / F-12), Glasgow Minimun Essential Medium (Glasgow MEM).

In this medium, ascorbic acid required for collagen synthesis is essentially added and other growth factors or differentiation inducing factors such as FGF (Fibroblast Growth Factor), HGF (Hepatocyte Growth Factor), IGF (Insulin-like Growth Factor), TGF Growth factor, VEGF, EGF, BMP, TNF, vitamin, interleukins, heparin, heparin derivatives, heparan sulfate, collagen, (Including insulin, trans-perine, aspartic acid, bovine serum albumin, linolenic acid), fibronectin, fibrin, platelet plasma, progesterone, serenite, B27-supplement, N2-supplement, ITS- Dexamethasone, sodium biluvurate, proline, L-glutamine and the like are added as needed. If necessary, antibiotics such as additives, antibiotics and antifungal agents for adjusting the acid bases such as HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), 10% FBS Fetal Bovine Serum) or 10% or more FBS when higher proliferation is desired, and 10% to 10% of the above-mentioned self-serum of the patient to facilitate clinical application.

In the present invention, the chondrocyte, cartilage precursor cell, synovial membrane-derived cell, synovial stem cell, osteoblast, bone marrow-derived cell, adult stem cell or mesenchymal stem cell derived from various tissues, adipose derived cell, (IPS cells) that have pluripotent and proliferative capacity similar to ES cells, such as stem cells (ES cells) and inducible pluripotent stem cells (iPS cells), in which various kinds of differentiated cells are initialized, Each cell material that can be treated by transplantation into the affected area is amplified by repeated subculture for about 2 weeks.

The chondrocytes showing high differentiation are 2 to 20 times or more, the pluripotent cells having the pluripotent and proliferative capacity similar to the ES cells and the differentiation ability for the chondrocyte cell are 20 to 100 times or more amplifiable. It is preferable to use a medium adjusted with 10% FBS, ascorbic acid in any cell growth medium.

When the confluency reaches about 90% while culturing, the amplified chondrocytes are removed by treatment with trypsin / EDTA and subcultured in a new medium. In the present invention, it is preferable that the subculture is limited to 3 times or less. This is because the cartilage cells are repeatedly passed through the cell, and degenerated (de-defferentiation) occurs more than 4 times, This is because the type of collagen that is the outer matrix changes from fibroblast-like to fibroblast-like from Ⅱ to Ⅰ.

The subculture of the chondrocyte and chondrocyte precursor cells of the present invention is performed by seeding the cells at a high density. Cell density at the time of sowing of said chondrocytes and cartilage precursor cells according to the present invention varies depending on the cultured cells, and 5000 / cm ² or more are preferred, and 10,000 / cm ² or more, and more preferably, 20,000 / cm < ² > or more. The density of regenerating cartilage tissue and bone tissue is more effectively regulated (2.0 +/- 0.4) x 10 < ⁴ > / cm < ² & ² or less, the cultured cells tend to change to the above fibroblast cells, that is, in the case of chondrocytes, the degree of the expression of the transcript is decreased and the object of the present technology can not be achieved

The cell spleen for transplantation therapy of the present invention is characterized in that the mixed cell complex is expressed as a cartilage sheath tissue. Expression of a trait as a cartilage sheath expresses a differentiation trait such as expressing a genetic trait such as SOX9 or HAS or a collagen type II involved in formation of extracellular matrix. Under the above conditions, chondrocyte cells can be amplified to a minimum of 2 to 20 times or more by culturing for about 2 weeks (i.e., time required up to 4 times of passage).

In addition, in addition to 10% FBS, ascorbic acid-heat-inactivated medium which is inactivated by heat in any cell growth medium known in the art, other proliferative differentiation factors are required Can be amplified to a degree above the degree of propagation.

In the step (3) of the production method according to the present invention, the cell material suspension in which one or two or more kinds of cells are singly or mixed in a culture medium is prepared, and the cells are maintained in a high density floating state, To prepare a mixed cell complex.

In this step, the mixed cell complexes need to add an excess of cell material that is capable of adhering cell materials sufficiently to form a cell spleen for transplantation therapy. Cells are originally produced by cells themselves and have a property of trying to adhere to each other by various kinds of adhesion factors, electrical attraction, chemical bonding, etc. which are expressed on the cell surface. Such properties are different depending on the type of each cell. With this nature, accompanied by a shaking motion, it artificially creates opportunities for cell-cell contact. Due to the flow of the culture fluid resulting from the continuous and repetitive shaking motion resulting from this artificial operation, each cell does not stagnate at the bottom of the culture vessel and the suspended state in the culture medium can be maintained during the shaking culture period.

Thus, a number of different cell materials rapidly attach to a small sized mixed cell complex that becomes a cell spoloid at the initial stage of shaking, and cell spoil for transplantation is grown. In particular, the cells expressing the chondrocyte and cartilage sheath tissues become more ecologically similar to the original cartilage cells while maintaining their size when they are present at a high density. In other words, in an environment suitable for maintaining the differentiation degree of cartilage tissue as normal cartilage, chondrocytes produce and secrete collagen type Ⅱ which is intrinsic extracellular matrix, resulting in relatively few cells as in the case of being present in the body, The outer matrix is similar to the abundant normal cartilage.

In the step (3), it is possible to use each medium described in the step (2) of the production method according to the present invention. The shaking culture was carried out in an environment in which the shaking intensity was set at 60 to 80 rpm using a shaker for shaking culture capable of moving in a three-dimensional 8-figure, a flat 8-figure, a planar circle, It is preferable to proceed. In particular, the incubation period is 1 to 7 days, and each cell material sufficiently amplified during the subculture period can shorten the incubation period compared to the existing autologous chondrocyte transplantation requiring cell culture period of 4 weeks or more As a result, it becomes possible to manufacture cell spleoids for transplant treatment in a short period of time. The shortening of this period can be established by a technique that can be applied to patients with elderly patients and patients with a wide range of injuries and from the viewpoint that they can contribute to alleviation of medical expenses due to shortening of the hospitalization period of patients, The total production time of the above-mentioned transplantation cell spleoid including the subculture period and the shaking culture period is preferably about 14 days. As a culture container, a non-adhesive culture dish designed to prevent cells from adhering to the surface thereof (i.e., a floating culture dish) or a spinner flask in the case of mass culture is used. For example, a HydroCell ™ culture dish (Cellseed, Co.), which exhibits hydrophilicity at about 37 ° C and does not allow any cell adhesion, can be used. Through this, it is possible to prepare a cell spleroid for implantation therapy with a size of about 10 mu m to 1,500 mu m.

In addition, the shake culture is applied by adjusting the cell density of the cell material suspension from 1.0 x 10 ⁴ / ml to 3,000 x 10 ⁴ cells / ml. However, when selecting an appropriate cell density, the kind of cell material used, The cell density of the cell material suspension within the above-mentioned appropriate range may be determined according to the bottom diameter, space, volume, size, etc. of the culture container during shake culture, if necessary. Further, when setting the intensity of the shaking culture and the mode of shaking, it is necessary to determine the shaking intensity and the mode of shaking according to the type of the cell material, the properties of the culture substrate and the bottom diameter, space, volume, The intensity of the shaking within the above range may be set. Depending on the need and intended purpose, the mode of shaking may be varied as the form of the motion and the intensity of the shaking progress over time,

In the step (3), the micro-carrier is a molecule for promoting the mutual adhesion of cells in the cell material suspension, wherein the micro-carrier has good mutual adhesion between the cells and the microcarriers do. The use of this microcarrier makes it possible to achieve good mutual adhesion between shake cell lyocell materials, enhance the efficiency of cell spoloid formation, and enhance expression of the trait with cartilage tissues suitable for transplantation. The microcarriers may be used singly or in combination of two or more when necessary. When two or more types are used in combination, different microcarriers may act as new microcarriers reacting with each other and exhibiting more beneficial properties. For example, among the following substances, electrocharacterically different properties of Fragmin (Dalteparin sodium) and Protamine sulfate form microcarriers, and the new microcarriers thus formed (Nakamura et al., J Biomed Mater Res A., May-12, Epub ahead of print (2009)) between the cells and the microcarriers.

The microcarriers may be selected from collagen, collagen derivatives, hyaluronic acid, hyaluronic acid derivatives, lubrication, mucin, chitosan, chitosan derivatives, polyrotakiic acid, polyrotakiic acid derivatives, chitin, (Dalteparin sodium), Protamine sulfate, Avidin, and the like), chitin derivatives, urethane, cellulose, agarose, gelatin, fibrinogen, fibrin, platelet platelet, heparin, heparin derivatives, Fragmin , Streptavidin, Biotin, Laminin, 2-Octyl Cyanoacryleate, Calcium Alginate, Polyglycerin, Polyethylene Glycol, Polyvinylpyrrolidone, Polyvinyl Alcohol, Polypropylene, Polyglycolic Acid, Polycaprolactam, One or two or more biocompatible materials selected from the group consisting of polymers, polylactic acid polycaprolactam copolymers and polyglycolic acid polycaprolactam copolymers, or bioabsorbable materials It is formed from a material. In addition, the microcarriers can be effectively used clinically, in view of the fact that substantially usable mixed cells can easily be used as a therapeutic method for producing and using the cellulase treatment spleen as a diaper, And an application is preferably obtained.

In the step (3), the shaking culture will be described with reference to Figs. 1, 2 and 3. Cellular material suspensions obtained by singly or in combination of one or more kinds of cells (cell material A and cell material B of FIG. 2) in a culture medium are prepared and cultured under shaking to adhere the cell materials to produce a mixed cell complex A second cell, which is a mixed cell complex in which first cell spoil, which is a product of the first shaking culture, and cell material are adhered to each other by performing shaking culture with only one process, or shaking culture of two or more times, It is possible to prepare new mixed cell complexes with spheroids. The cell material suspension prepared by mixing the cells in the culture medium with the primary cell spleoid as the center is further prepared and then subjected to the second shake culture, whereby the primary cell spleod, which is the result of the first shake culture, In a process for preparing a new mixed cell complex with a secondary cell spoloid which is a mixed cell complex in which materials are bonded to each other, a state immediately after the introduction of the cell material of the primary cell spoil and the second shaking culture is examined under a phase contrast microscope And the results are shown in Fig.

By shaking culture more than two times, the structure of the primary cell spoloid, the resultant product, can be artificially altered. For example, if the cell material of the secondary cell spoiloid nearer to the surface layer is chondrocytes and the cell material of the primary spoil located at the deeper side is used as the synovial cell, a synovial derived cell with a high proliferation rate The formation of secondary cell spoiloids, even with only a small number of cell materials within a limited culture period, is believed to be the result of the formation of primary cell spoiloids, which occupy most of the lloids, It becomes sufficient. The production ability considering the type of the cell material and the type and structure of the cell material in the shake culture of the above plural stages may be selected by comprehensively judging the proliferative capacity of each cell material and the anatomical and histological developmental aspects in vivo.

In the manufacturing method step (4) according to the present invention, the cell spoiloid prepared for cell transplantation treatment is isolated and transplanted into the damaged lesion. In the above step (3), if shaking culture is performed for 1 to 7 days, various sizes of implantable cell spleens are present in the culture dish. In order to separate them by size or to separate them by size, the cells for sterilization were inhaled into a new culture dish (suspension culture dish) with a micropipette while observing under a phase difference microscope using a sterilized micropipette as an embodiment Method can be used. At this time, the culture dish is circularly moved, and the centrifugal force is used to collect the cell spoil for transplantation therapy. The tip of the micropipette is used to culture the culture medium used for the transplantation cell spoloid, Inhalation of a solution having a composition similar to that of a body fluid such as a suspension, a microcarrier-containing solution, various buffers, and physiological saline, etc., can absorb the implantable therapeutic spore of a size suitable for the size of the tip size. In a similar manner, using a needle and a syringe may be used to prepare a sponge for graft treatment in a form that can be used for endoscopic or arthroscopic or arthroscopic surgery in the process of transplanting a damaged lesion.

In the step (3), when the prepared cell spoloid is transplanted to place the cell spoil on the transplantation site, which is a damaged part, the cell spoloid may be different depending on the cell constituting the surface of the cell spoloid. Has a property of intending to adhere to each other by various kinds of adhesion factors, electrical attraction, chemical bonding or the like which is produced by the cell itself and is expressed on the surface of the cell, and the cell spoloid, However, the microcarriers may be used when the mixed cell complex is desired to be better immobilized to the damaged lesion and transplanted within a short period of time, and to quickly bind the patient and the cell spleenoid. The microcrystalline may be selected from the group consisting of collagen, collagen derivatives, hyaluronic acid, hyaluronic acid derivatives, lubrinic acid, mucin, chitosan, chitosan derivatives, polyrotakiic acid, polyrotakiic acid derivatives, chitin, chitin derivatives Dalteparin sodium, Protamine sulfate, Avidin, Streptavidin, Fibrin, Heparin, Heparin derivatives, Fragmin, Dalteparin sodium, Fibrin, , Biotin, laminin, 2-octyl cyanoacryleate, calcium alginate, polyglyceric acid, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polypropylene, polyglycolic acid, polycaprolactam, polylactic acid polyglycol acid polymer, One or two or more biocompatible or bioabsorbable materials selected from the group consisting of polylactic acid polycaprolactam copolymer and polyglycolic acid polycaprolactam copolymer And may be selected from the group of microcarriers described above in consideration of the properties of the microcarriers, the composition of the cell spleen for transplantation therapy, the environment of the transplantation site, and the like.

On the other hand, in consideration of the shape of the injured area and the surrounding area, the microcarrier alone or separately used for fixing the mixed cell complex to a damaged area satisfactorily, Derived stem cells, mesenchymal stem cells, adipose derived cells, adipose derived stem cells, embryonic stem cells, osteoblast-derived stem cells, mesenchymal stem cells, osteoblast-derived cells, (ES cells), inducible pluripotent stem cells (iPS cells) that have been resynchronized with various kinds of differentiated cells, and the like, which have pluripotency and proliferative capacity similar to ES cells and have differentiation ability for chondrocyte cells, One or two or more kinds of cells capable of exhibiting a therapeutic effect by being transplanted into a cell can be cultured for a certain period of time, A collagen, a collagen derivative, a hyaluronic acid, a hyaluronic acid derivative, a lubrication, a lubrication, a mucin, a chitosan, a chitosan derivative, a polyrotactic acid, (Dalteparin sodium), cholinesterase inhibitors, cholinesterase inhibitors, cholinesterase inhibitors, cholinesterase inhibitors, cholinesterase inhibitors, cholinesterase inhibitors, cholinesterase inhibitors, It is also possible to use an acid such as Protamine sulfate, Avidin, Streptavidin, Biotin, Laminin, 2-Octyl Cyanoacryleate, Calcium Alginate, Polyglyceric acid, Polyethylene glycol, Polyvinylpyrrolidone, Polyvinyl alcohol, Polypropylene, Polyglycolic acid, Poly One or two selected from the group consisting of caprolactam, polylactic acid polyglycol acid copolymer, polylactic acid polycaprolactam copolymer and polyglycolic acid polycaprolactam copolymer Or more bio-compatible material or a bioabsorbable material, may be coated or filled with an auxiliary in cartilage or bone tissue with respect to the surface of the artificial material's bio-sheet structure, and various shapes of the structures formed by. Here, FIG. 4 shows an example in which the mixed cell complex is supplementarily used with a sheet-like structure so as to fix the mixed cell complex to a damaged part well.

In the present invention, cell transplantation is a cell component of a cell spoloid for transplantation therapy of a mixed cell complex, but it is preferable that the cell spoloid is formed by a molecule secreted from constitutive cells and a produced extracellular matrix, A carrier solution having a similar composition to a body fluid such as a culture medium and a cell suspension used, a microcarrier-containing liquid or a microcarrier-containing liquid, various buffers, and physiological saline can be transplanted alone or simultaneously into the damaged lesion for treatment, It is possible to perform cell transplantation therapy which can exert its original function in a state of being expressed as a transcript.

According to another aspect of the present invention, the cell spleen for transplantation therapy may be used as a system for evaluating the pharmacological effects of toxic substances, chemicals, physiological actions, and toxicity of compounds, medicines, and toxic substances separately from the purpose of treatment. The use of the above cell implant is not particularly limited. In addition to the purpose of treating various diseases, all tests, studies, and identification of material properties using cell spheroids can be carried out using cell spoiloids . For example, spleens prepared from the various adult stem cells obtained by the method of the present invention can be used in combination with IGF, TGF, TRANSFERIN, insulin, FBS, GA-1000, ITS-supplement, dexamethasone, ascorbic acid, It is also possible to induce differentiation into chondrocytes by treating factors such as acid, sodium, and proline. By transplanting the differentiated cells obtained through this operation into a patient, autologous cell transplantation through stem cell therapy using stem cells becomes possible One cell spoloid can be used to directly target the produced cell spoiloid to the drug efficacy or toxicity of a test substance involved in cartilage regeneration. The use of the cell spleoid for transplantation therapy of the present invention is not limited by the above specific embodiments.

According to another aspect of the present invention, there is provided a method for cultivating a cell material comprising culturing a cell with a primary culture, which is the product of the first shake culture, A second mixed cell complex, which is a mixed cell complex in which primary cell spoil, which is a product of the first shaking culture, and cell materials are adhered to each other, is prepared by preparing a cloudy solution, . &Lt; / RTI > When multiple stages of shaking culture having such characteristics are carried out, the structure of the cell spoloids can be formed into a multilayered layered shape. For example, when the knee cartilage tissue is observed from an anatomical, histological, or embryological point of view, the shape of cartilage cells constituting the knee cartilage tissue changes from the surface layer toward the bone marrow toward the deeper side. The cartilage cells differ in their shape and function from each other. It is possible to construct in vitro the microenvironment which is very similar to the in vivo environment by producing the cell spoloid structure from cells which are different from each other in this layered structure. By evaluating the physiological actions and toxicity of compounds, medicines, toxic substances, and the like by the above system using the cell spoiloid thus prepared, it is possible to evaluate and predict more accurately.

The further enhancement of the inherent specific function of the cell matrix is that, when a large group of cells is preserved under extracorporeal conditions, the group of these cells, due to gravity in general, is loosened, The culture medium maintained in a high density floating state provided by the manufacturing method according to the present invention enhances mutual adhesion in a group of cells and can be closely contacted with each constituent cell to communicate with each constituent cell, Promoting the production of extracellular matrix, thereby enhancing the inherent specific function.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to be illustrative of the present invention and are not to be construed to limit the present invention. It will be apparent to those skilled in the art that the present invention can be carried out without departing from the scope of the present invention.

[Example 1]

Preparation of Cellular Spheroids for Combination Therapy with Mixed Cell Complexes Mixed with Rabbit Knee Joint Cartilage Cells and Synovial Cells and Cartilage Depletion Model

1-A. Isolation and culture of chondrocytes and synovial cells

Cartilage and synovial tissues were collected from the knee from a rabbit (Japanese White Rabbit, 1 kg ± 200 g, female) to remove skin tissue, subcutaneous tissue, muscle tissue, subchondral bone, ligament, meniscus and other connective tissues Then, it was placed in a commercially available 50 ml centrifuge container, and then chopped using surgical scissors and finely sliced. 1% antibiotic / antifungal agent was added to D-MEM / F-12 medium (Gibco. Co.) with 1.25% trypsin (Invitrogen Co.) and 0.5% collagenase class I (Worthington Biochemical Co.) Were dissolved in a medium adjusted with a mixture (10,000 units / ml of penicillin G, 10,000 占 퐂 / ml of streptomycin, 25 占 퐂 / ml of amphotericin, Gibco Co.), and the primary digestive enzyme and the second digestive enzyme solution Prepared.

The cartilage tissue and synovial tissue were placed in an aseptic glass container and sterilized magnet bar for electrostatic sterilization was put in each of the above digestive solutions. Then, in a conventional incubator for cell culture, conditions of 37 ° C and 5% CO ₂ (Model: Magnetic Stirrer RCN-3D, EYELA Co.) under the conditions of 60 rpm.

At this time, the chondrocytes and synovial tissues were separated into single cells by 1 hour treatment with primary digestive enzymes and additional 3 hours treatment with secondary digestive enzymes. The cell suspension thus obtained was passed through a cell strainer (BD Falcon ™: BD Biosciences) made of nylon of 70 μm and 40 μm, and washed twice with phosphate buffer solution.

Single cells obtained through this procedure were cultured inoculated on a growth medium for amplifying the number of chondrocytes and synovial cells. Cell density during the first incubation sowing in the case of cartilage cells (2.0 ± 0.4) × 10 with ⁴ / cm ^2, synovial-derived cells (1.0 ± 0.4) × 10 with ⁴ / cm ² and, if the time since the sub-cultured chondrocytes If the cell density is ^{(2.0 ± 0.4) × 10 4} / cm ² to, synovial-derived cells was set to ^{(1.0 ± 0.4) × 10 4} / cm 2. To the D-MEM / F-12 medium (Gibco. Co.) was added 10% heat inactivated FBS, 1% antibiotic / antifungal mixture (10,000 units / ml penicillin G, 10,000 ㎍ / ml streptomycin , 25 μg / ml amphotericin, Gibco. Co.) and 50 μg / ml ascorbic acid. When the confluency reached 90%, the cells were treated with trypsin-EDTA Chondrocytes and synovial cells were detached from the culture dish.

In the subculture period for cell expansion for 12 to 13 days, chondrocytes were transfected twice and synovial-derived cells were transfected up to 4 times. Finally, the cell material for preparing the cell spoloid for transplantation treatment of the mixed cell complex , Chondrocytes of the third generation and synovial cells of the fourth and fifth generation are used.

1-B. Tagging of isolated cultured chondrocytes and synovial cells

Each isolated cultured chondrocyte and synovial membrane-derived cells recovered through Example 1-A were cultured in D-MEM / F-12 medium (Gibco. Co.) with 1% antibiotic / antifungal mixture (10,000 units / ml penicillin (1,800 rpm / 5 min, 24 ° C), and then cultured in a medium supplemented with chondrocyte and chondrocyte Each cell of the synovial membrane-derived cells was examined under a fluorescence microscope or a confocal laser microscope

Time) using a PKH staining kit (Sigma Co.). The cartilage cells were stained with a fluorescent red tag MINI26 and the synovial cells were stained with fluorescence staining using the MINI67 kit (Fluorescent Cell Linker Mini Kit for General Cell) And synovial cells were compared with each other. By this work, cell suspensions of tigated chondrocytes and synovial cells derived from a fluorescent reagent were prepared.

1-C. Preparation of mixed cell complex by shake culture

The cell suspensions of cartilage cells and synovial membrane-derived cells, which were subjected to appropriate number of subcultures and fluorescence reagent-tagging, were subjected to D-MEM / F-12 medium (Gibco. Co.) (10,000 units / ml penicillin G, 10,000 占 퐂 / ml streptomycin, 25 占 퐂 / ml amphotericin, Gibco. Co.) and 10% FBS inactivated thermally, 1% antibiotics / Resuspended in 5 ml using a medium adjusted with 50 / / ml of ascorbic acid, and chondrocyte and synovial membrane-derived cells alone or in combination with chondrocytes and synovial membrane-derived cells at the same rate (i) 100% synovial cells, 0% cartilage Cell suspension of cell constitution, 75% synovial cell, 25% cell suspension, 50% synovial cell, 50% cell suspension, 25% synovial cell, 75% cell suspension , ⑤ 0% synovial cells / 100% chondrocytic cell suspension) They were prepared as single or mixed cell suspension. As a culture container, a shaker for shaking culture (model: Double Saker NR (trade name), manufactured by Cellseed Co., Ltd.) under the conditions of 37 ° C and 5% CO ₂ in a conventional cell culture incubator using a 60 mm HydroCell ™ culture dish -3, TAITEK Co.) for 1 to 5 days while adding a planar circular movement at 70 rpm.

The time required for the total preparation of the cell spleen for transplantation therapy, including subculture and shake culture, was about 14 days. Here, an example of a supplementary use of a sheet-like structure (chondrocyte sheet) to fix the mixed cell complex to a damaged region well is shown as an example, and the manufacturing process schedule is shown in FIG.

The cell spoloids obtained at the elapsed time of the incubation period were observed by adding a filter that selectively passes only the wavelength range suitable for the phase contrast microscope under observation of the chondrocytes and the synovial cells derived from the tethered cells or the fluorescence reagent The results are shown in Figs. 6 and 7, respectively. FIG. 6A shows the ratio of chondrocyte and synovial cell-derived cells to the cell suspension of 75% synovial membrane-derived cells and 25% chondrocytic cell culture immediately after commencement of shaking culture, B: 12 hours, C: 24 hours, D Are the results observed under a phase contrast microscope after elapse of 36 hours. FIG. 7A shows the ratio of chondrocyte and synovial cell-derived cells. ④ Cell suspension of 25% synovial membrane-derived cells and 75% chondrocytic cell culture was subjected to shaking culture immediately after 12 hours, after 24 hours, And a filter for selectively passing only a wavelength within a suitable range in the phase difference microscope after elapsed time was observed.

In addition, shake culture was carried out with the chondrocyte and synovial membrane-derived cells solely or with the chondrocyte and synovial membrane-derived cells at the same rate as that of the cell suspension, The shaking culture was carried out for 4 days while adding exercise, and the results are shown in Figs. 8A and 8B. 9 is a graph showing the results obtained by adding a filter that selectively passes only a suitable range of wavelengths under the condition of a phase difference microscope 36 hours after the commencement of the shake culture on the cell suspension of chondrocytes and synovial membrane- Respectively. Under confocal laser microscope, the cells were subjected to shake culture for 5 days in the ratio of chondrocytes and synovial cells, ③ 50% synovial cells, and 50% chondrocytic cell suspensions. The results are shown in FIG.

Each cell material constituting the cell spleen for transplantation therapy is not stagnant at the bottom of the culture container due to the flow of the culture fluid caused by the continuous repetitive motion resulting from an artificial operation with the lapse of time, A large number of other cell materials are rapidly adhered to the floating suspension state in a small size mixed cell complex which is maintained during the shaking culture period and made into the cell spoil of the initial stage of the shaking culture, Respectively. During this period, the mixed cell complexes began to be visually observable after 12 hours from the start of the shaking culture, and were observable throughout the course of the shaking culture. The results of visual observation of the obtained cell spoloids are shown in Fig.

The mixed cell complexes were confirmed to have a smooth outline with time. The results observed under a phase contrast microscope after 125 hours of incubation start are shown in FIGS. 6 and 7, respectively.

The diameter of the small cell spoloid was about 250 ± 100 ㎛, and the diameter of the large cell spoloid was about 700 ± 250 ㎛.

1-D. Histopathological examination

The ratio of chondrocytes and synovial cells derived from Example 1-C (3) Cell suspension of 50% synovial cells and 50% chondrocytic cells were cultured for 5 days, and cell spheroids were cultured in 4% paraformaldehyde The solution was fixed with 15% sucrose solution, 30% sucrose solution, and frozen sections were prepared. The sections were cut and prepared for histopathological examination. Biotin-peroxidase complex technique (LSAB 2 kit / HRP, DAKO JapanCo.) Was performed using 3-amino-9-ethylcarbazole (AEC) substrate-chromogen solution (DAKO Japan Co., Ltd.). Immunohistochemical staining for collagen type Ⅱ was performed according to the protocol of the manufacturer. The test results are shown in Fig. As a result, the presence of the strongly stained collagen type II was confirmed, which means that the cells and tissues of the stained area were expressed as a cartilage tissue similar to a normal or healthy state in the body.

1-E. Transplantation of cell spheroids into rabbit knee full-layer injury model

Ratio of cartilage cells and synovial membrane-derived cells obtained through Example 1-C (2) Cell suspensions obtained by subjecting cell suspensions of 75% synovial membrane-derived cells and 25% cartilage cells to shake culture were obtained by micropipette, A 5 mm diameter defect in the knee cartilage of the femur located below the patella of the rabbit (Japanese White Rabbit, 3 kg ± 500 g, female) was created by moving the cartilage defect from the cartilage surface layer to the cartilaginous bone and moving it to the damaged area 14A). Four weeks after the transplantation, the femur was sacrificed at the sacrifice of the cell sphere and fixed with 4% paraformaldehyde solution. After demineralization, the paraffin section was cut and the section was cut for histopathological examination Prepared. Biotin-peroxidase complex technique (LSAB 2 kit / HRP, DAKO JapanCo.) Was performed using 3-amino-9-ethylcarbazole (AEC) substrate-chromogen solution (DAKO Japan Co., Ltd.). Immunohistochemical staining for collagen type Ⅱ was performed according to the protocol of the manufacturer. Fig. 14A shows the results of the gross observation after transplantation, in which a defect of 5 mm in diameter was formed in the knee cartilage of the femur side under the patella of the rabbit, . Histopathological examination is also shown in Figure 14B. As a result, the presence of the deeply stained collagen type II was confirmed, indicating that the cells and tissues of the stained area 4 weeks after transplantation in the environment of the transplanted cell spoloid were similar to the normal or healthy state of cartilage tissue &Lt; / RTI >

[Example 2]

Preparation of primary cell spleoids from pancreatic endocrine cells (PEC) isolated from rat islet cells into single cells

2-A. Isolation and Culture of PEC

The pancreas was extracted from rats (Brown Norway Rat or Lewis Rat, 350 ± 50 g) and added to a solution of 10% FBS and HEPES in Hanks' Balanced Salt Solutions (HBSS) (Gibco.Co. Co.) was dissolved to make 2 mg / ml of collagenase P. The collagenase P was digested and purified by concentration gradient method using Histopaque (Sigma Co.) and recovered.

The isolated islets are treated with trypsin / EDTA for a short time of about 5 minutes and isolated by PEC. The PEC thus isolated is inoculated into a growth medium for quantitative amplification of the cells isolated by the same method as in 1-A of Example 1 and cultured. The amplified PEC is removed and subcultured in a fresh medium. The composition of the medium described in 1-A of Example 1, the type of digestive enzymes and culture dishes used, the cell density at seeding at the time of initial cultivation, and the cell density at the time of subculturing were suitably modified according to the intended use .

2-B. Preparation of mixed cell complex (primary cell spoloid) by shaking culture

Each isolated culture PEC obtained through Example 2-A was recovered by subculture and the recovered PEC was cultured in D-MEM / F-12 medium (Gibco. Co.) with 10% FBS heat inactivated, 1 The culture medium was adjusted with an antibiotic / antifungal mixture (10,000 units / ml penicillin G, 10,000 占 퐂 / ml streptomycin, 25 占 퐂 / ml amphotericin, Gibco. Co.) and 50 占 퐂 / ml ascorbic acid And resuspended in 5 ml to prepare a cell suspension. As a culture container, a shaker for shaking culture (model: Double Saker NR (trade name), manufactured by Cellseed Co., Ltd.) under the conditions of 37 ° C and 5% CO ₂ in a conventional cell culture incubator using a 60 mm HydroCell ™ culture dish -3, TAITEK Co.) for 1 to 5 days while adding a planar circular movement at 70 rpm.

Each cell material constituting the primary cell spoloid is not stagnant at the bottom of the culture container due to the flow of the culture fluid generated by the continuous repetitive motion resulting from an artificial operation with the lapse of time, A large number of other cell materials are rapidly adhered to the floating suspension state in a small size mixed cell complex which is maintained during the shaking culture period and made into the cell spoil of the initial stage of the shaking culture, Respectively. During this period, the mixed cell complexes began to be visually observable after 12 hours from the start of the shaking culture, and were observable throughout the course of the shaking culture.

The primary cell spoloids were found to have a smooth outline over time.

2-C. Use of Primary Cellular Spheroids

A cell material suspension in which one or more kinds of cells were separately or mixed in the culture medium was prepared based on the primary cell spleoid composed of the PECs obtained in Examples 2-A and 2-B, and cultured in two or more shaking cultures And a second mixed cell complex, which is a mixed cell complex obtained by mutually adhering the primary cell spoil, which is the product of the first shaking culture, with the cell material, is repeated twice or more Shake culture.

The present invention relates to a cell-spleen for transplantation therapy, which is a mixed cell complex, which is a tissue similar to a normal or healthy state in the body, a cell spleen which is a collection of a plurality of cells capable of replacing various tissues constituting the body expressing the trait And each cell material constituting the mixed cell complex is separately prepared, each prepared cell is subcultured to be amplified, and one or two or more kinds of cells are separately or mixed in the culture medium to prepare a cell material suspending liquid. The cell material is mutually adhered to each other by shaking culture to prepare a mixed cell complex, and the cell spoloids prepared for the cell transplantation treatment are separated and transferred to a predetermined place according to each purpose of use. The method of manufacturing Lloyd, the method of using Lloyd, and the physiological effects of drugs and toxic substances Or cytotoxicity of a bone marrow, wherein each cell material constituting the cell spoil for transplantation therapy is applied to an autologous chondrocyte cell implant to treat damage to cartilage tissue and bone tissue, The present invention relates to a transplantable cell for transplantation, which is expressed as a cartilaginous tissue by using various stem cells or chondrocyte precursor cells which have not only a single use but also one or two or more kinds of high proliferative ability and easy differentiation ability into cartilage tissue. It is possible to prepare a large amount of spheroids, and it is also possible to considerably improve the technique of treatment for a wide range of cartilage tissues and bone degeneration or elongation of the elderly, degeneration of cartilage or degeneration of cartilage tissues and partial depletion of cartilage tissues, .

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing a mixed cell complex in which a cell material is adhered to each other by preparing a single cell material suspension in a culture medium and cultivating the shake in a culture according to an embodiment of the present invention.

FIG. 2 is a schematic diagram showing a mixed cell complex in which two kinds of cells, cell material A and cell material B, are prepared by preparing a mixed cell material suspension in a culture medium and shaking culture, .

Fig. 3 is a graph showing, in another aspect of the present invention, a method for culturing shake cultures of a plurality of stages carried out twice or more times, wherein one or two or more cells are cultured in a culture medium A mixed cell complex in which primary cell spoil, which is a product of the first shaking culture, and cell material are adhered to each other, is prepared by preparing a cell material suspension of a single cell or a mixed cell material in the cell culture medium, Lt; RTI ID = 0.0 > a < / RTI > mixed cell complex.

FIG. 4 is a schematic view showing an embodiment of a cell spoil for transplantation, which is a mixed cell complex of a therapeutic method supplementarily used with a sheet-like structure (cartilage cell sheet) in an aspect of the present invention .

FIG. 5 illustrates an embodiment of the present invention in which the mixed cell complex is supplementarily used with a sheet-like structure (chondrocyte cell sheet) so as to be favorably fixed at a predetermined place according to the method of use for each purpose, An example of a manufacturing process schedules with a total production time of about 14 days for a cell spleod for transplantation therapy, including a subculture period and a shaking culture period, is shown.

FIG. 6 is a graph showing that, in one embodiment of the present invention, a mixed cell complex in which cell materials are adhered to each other by shaking culture of a cell suspension having a ratio of chondrocytes and synovial cells to a cell suspension having a composition ratio of 75% The results are shown under a microscope.

FIG. 7 is a graph showing the results of experiments in which the cell mixture is adhered to each other by shaking culture of a 25% synovial cell-derived cell and a 75% chondrocytic cell suspension in a ratio of chondrocyte and synovial cell derived from a synovial cell to a phase contrast microscope And the results are shown in FIG.

FIG. 8A is a diagram illustrating an example of an embodiment of the present invention. In FIG. 8A, the cell materials are mutually adhered to each other by shaking culture with one kind of cells (that is, a cell suspension of ⑤ 0% synoviocyte- Were observed under a phase contrast microscope and the results are shown.

FIG. 8B is a graph showing the results of cell culture of a cell suspension (a cell suspension of 5% 0% synovial cells, 100% chondrocytes) The mixed cell complexes were observed under a phase contrast microscope and the results are shown.

FIG. 9 is a graph showing the ratio of chondrocytes and synovial cells derived from a 100% synovial cell, a 0% chondrocytic cell suspension, a 75% synovial cell, a 25% chondrocyte cell suspension, Cell suspension of 50% synovial membrane-derived cells, 50% chondrocytic cell suspension, cell suspension of 25% synovial membrane-derived cells, cell suspension of 75% chondrocyte composition, cell suspension of 0% synovial membrane-derived cells, The results are shown in the result of observing a mixed cell complex in which cell materials are mutually adhered under a phase contrast microscope.

10 shows, in an embodiment of the present invention, a mixed cell complex in which cell materials are adhered to each other by shaking for 5 days in a cell suspension having chondrocyte and synovial cell-derived ratio of ③50% synovial cells and 50% Were observed under a confocal laser microscope and the results are shown.

Fig. 11 is a graph showing the results of shake culturing performed in two or more different shaking cultures. Fig. 11 is a graph showing the results of the first shake culture, A mixed cell complex in which primary cell spoil, which is a product of the first shaking culture, and cell material are adhered to each other, is prepared by preparing a cell material suspension of a single cell or a mixed cell material in the cell culture medium, The result of observing the mixed cell complex formed with lyop under a phase contrast microscope is shown.

Figure 12 shows that, in another embodiment of the present invention, the mixed cell complex started to be visually observable after 12 hours from the start of the shaking culture and could be observed in the entire course of the shaking culture. The results of gross observations of the obtained cell spoloids are shown.

FIG. 13 is a graph showing the results of a cell culture of 50% synovial cells and 50% chondrocytic cells in a ratio of chondrocytes and synovial cells derived from chondrocytes. The results of histopathological examination of immunohistochemical staining of the complex are shown.

14A is a cross-sectional view of a mixed cell complex in which a cell material is adhered to each other by shaking culture for 3 days in a cell suspension having a ratio of cartilage cells and synovial membrane-derived cells of 25% The results of gross visual observation after transplantation of the whole knee injured cartilage in the femur side of the rabbit were shown.

FIG. 14B is a graph showing the results of a mixed cell complex in which cell materials are adhered to each other by shaking for 3 days in a cell suspension having a ratio of chondrocyte and synovial membrane-derived cells of 25% The results of histopathological examination of immunohistochemical staining performed after 4 weeks of transplantation of the cell spleoids, which were transplanted to the injured area of the injured knee cartilage of the entire femur side of the rabbit, were transplanted.

Claims

Cell spleoids (mixed cell complexes) mixed with cartilage cells of the knee articular cartilage and synovial cells.

The method according to claim 1,

Wherein the mixed cell complex is a mixture of the knee joint cartilage cells and the synovial cells in a ratio of 1: 3 to 3: 1.

3. The method according to claim 1 or 2,

Wherein the mixed cell complex is composed of cells derived from autologous, allogeneic or heterogeneous tissues or two or more kinds of cells derived therefrom (mixed cell complex).

3. The method according to any one of claims 1 to 2,

Wherein the mixed cell complex has a size of 10 탆 to 1,500 탆.

A cell therapeutic composition for cartilage regeneration comprising the cell spoloid (mixed cell complex) of claim 1 as an active ingredient.

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